Liang et al. Virology Journal (2015) 12:49
DOI 10.1186/s12985-015-0275-7
RESEARCH
Open Access
ATP synthesis is active on the cell surface of the
shrimp Litopenaeus vannamei and is suppressed
by WSSV infection
Yan Liang, Meng-Lin Xu, Xiao-Wen Wang, Xiao-Xiao Gao, Jun-Jun Cheng, Chen Li and Jie Huang*
Abstract
Background: Over the past a few years, evidences indicate that adenosine triphosphate (ATP) is an energy source
for the binding, maturation, assembly, and budding process of many enveloped viruses. Our previous studies
suggest that the F1-ATP synthase beta subunit (ATPsyn β, BP53) of the shrimp Litopenaeus vannamei (L. vannamei)
might serve as a potential receptor for white spot syndrome virus (WSSV)’s infection.
Methods: BP53 was localized on the surface of shrimp hemocytes and gill epithelial cells by immunofluorescence
assay and immunogold labeling technique. Cell surface ATP synthesis was demonstrated by an in vitro bioluminescent
luciferase assay. Furthermore, the expression of bp53 after WSSV infection was investigated by RT-PCR test. In addition,
RNAi was developed to knock down endogenous bp53.
Results: BP53 is present on shrimp cell surface of hemocytes and gill epithelia. The synthesized ATP was detectable in
the extracellular supernatant by using a bioluminescence assay, and the production declined post WSSV binding and
infection. Knocking down endogenous bp53 resulted in a 50% mortality of L. vannamei.
Conclusion: These results suggested that BP53, presenting on cell surface, likely served as one of the receptors for
WSSV infection in shrimp. Correspondingly, WSSV appears to disturb the host energy metabolism through interacting
with host ATPsyn β during infection. This work firstly showed that host ATP production is required and consumed by
the WSSV for binding and proceeds with infection process.
Keywords: White spot syndrome virus, Shrimp, F1-ATP synthase beta subunit, Cell surface ATP synthesis, Virus binding,
RNAi, Receptor
Introduction
White spot syndrome virus (WSSV), the only member
of the genus Whispovirus of the family of Nimaviridae,
has emerged globally as one of the most prevalent and
lethal pathogen for Penaeid shrimp species since its first
outbreak in 1992 [1]. The better understanding of its
pathogenesis, especially the nature of virus–host interactions, will eventually lead to the development of new
strategies to control white spot viral disease. It is well
known that attachment/binding to the host cell surface
is essential for initiation of a viral infection [2]. This
virus–host interactions may also trigger a serial host
* Correspondence: huangjie@ysfri.ac.cn
Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of
Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of
Fishery Sciences, No.106 Nanjing Road, Qingdao 266071, China
immune responses against the invader as well as some
modifications of host gene expression to facilitate virus
replication [3]. Recently several WSSV envelope proteins
[VP37 (VP281), VP28, VP187, and VP53A], and some
cellular proteins of shrimp [PmRab7, β-integrin, PmCBP,
and F1-ATP synthase β subunit (ATPsyn β, also named
BP53)] have been reported in relation to the process of
virus particles attachment and entry into host cells [4-9].
However, it is still unclear about how viral infections
cause profound alterations in host cells [7].
ATPsyn β, as part of F1Fo ATP synthase complexes,
was originally described in the inner membrane of mitochondria. However, ATPsyn β have been found on the
surface of tumor cells, and serves as a receptor of
angiostatin [10]. In 2009, ATPsyn β was also reported on
the surface of Hpt cells from crayfish Pacifastacus
© 2015 Liang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
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unless otherwise stated.
Liang et al. Virology Journal (2015) 12:49
leniusculus (P. leniusculus). It was identified as a receptor for the invertebrate cytokine astakine and is involved
in hematopoiesis [10]. In our previous study, we identified an ATPsyn β (named as BP53, GenBank accession
number EU401720) in shrimp, which served as a receptor for WSSV binding [8]. Further study indicated that
the viral virulence can be attenuated after mixing the
WSSV with recombinant (r) BP53 [8]. This is the the
first work aimed to elucidate the role of shrimp ATPsyn
β with WSSV infection. In 2012, VP37, an important
structural protein of WSSV was also demonstrated to be
able to binding with ATPsyn β in another lab. Their results also revealed that three anti-ATPsyn β monoclonal
antibodies could partially block the binding of WSSV to
shrimp both in vitro and in vivo [11]. Based on these
studies, we speculate that BP53 might act as a candidate
receptor of WSSV.
The objectives of the present study were 1) to probe
whether BP53 is present on the hemocyte and gill cell
surface of shrimp as candidate receptor for WSSV infection, and 2) to investigate its biological function during
WSSV infection.
Results
BP53 polyclonal antibody preparation and specificity
characterization
After immunization of New Zealand rabbit with rBP53
protein, the antiserum was obtained. The western blot
results showed that the polyclonal antibodies could specifically identify BP53 in both recombinant cell lysates
and extracted proteins of gill membrane (Figure 1).
BP53 immunolocalization on shrimp hemocytes cell surface
The presence of the BP53 on the surface of hemocytes
was detected by immunofluorescence with the polyclonal
antibodies specific for the β subunit of ATP synthase generated against the recombinant BP53. The immunofluorescence was observed in each cell as one or more irregular
clusters of punctate structures (Figure 2A, B), suggesting
an organized distribution on the cell surface.
Figure 1 Specificity Characterization of the polyclonal antibody
against BP53 by western blot. Line marker, pre-stained protein
molecular mass markers (MBI, USA); Line 1 to Line 3, SDS-PAGE of gill
membrane proteins extracted from WSSV-free Litopenaeus vannamei
(Line 1), the lysates from E. coli Top10 cells with blank plasmid pBADgIIIB (Line 2, negative control), lysates of E. coli Top10 cells with
recombinant plasmid pBAD-gIIIB-BP53 (Line 3). Line 4 to Line 6,
identification of BP53 using anti-rBP53 antibody by western blot.
Samples loaded were as same as Line 1 to Line 3 in sequence.
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Fluorescence signal on cell surface of Figure 2A, B was
determined on the basis of two criteria. (i) Permeabilized
cells produced a dramatically different reticular pattern,
with a characteristic perinuclear distribution (Figure 2D).
(ii) Staining with a known cytoskeleton protein marker
actin, produced no detectable signals on intact cells
(Figure 2E), while actin staining appeared in cells with
enhanced permeability (Figure 2F). This result indicates
that anti-BP53 antibodies were binding to the extracellular component of BP53 on the cell surface.
BP53 immunolocalization on shrimp gill cuticular
membrane
For subcellular localization of BP53 in the gill tissue,
both immunofluorescent assay and immunogold assay
were performed. Punctate fluorescent signals were visible in the cuticular epithelium along the gill filament
(Figure 3A, B), a tissue that is most susceptible to
WSSV. Conversely, no signal was detected in the negative control group (samples incubated with pre-immune
serum only) (Figure 3C).
Immunogold assay with Au colloidal nanoparticles
conjugated antibodies on ultrathin section of L. vannamei gill tissue also showed even distribution of numerous gold nanoparticles along the cellular membrane of
gill tissue (Figure 3F), while no gold particles were observed in the negative control group (Figure 3G).
Cell surface ATP synthesis is active and being inhibited by
WSSV infection
The F1FO ATP synthase holoenzyme efficiently catalyzes
both the forward ATP synthase reaction and the reverse
ATP hydrolysis reaction. In order to detect ATP synthase activity on the cell surface, the ATP production in
the extracellular supernatant was measured using a bioluminescence assay. ATP generation was detected in the
presence of ADP and Pi substrates supplemented in the
external medium. ATP produced by WSSV-free cells is
4.36 ± 0.24 pmole/10 cells. In contrast, the ATP production was reduced to 42.9% in cells from WSSV infected
shrimp (1.87 ± 0.25 pmole/10 cells) (Figure 4). Further,
ATP concentration dropped to 86.7% (3.78 ± 0.14
pmole/10 cells) when the cells were incubated with
WSSV at 4°C for only 1 h. When rBP53 antibody was incubated with WSSV infected cells, the ATP production
was further decreased from 42.9% (1.87 ± 0.25 pmole/10
cells) to 30.5% (1.33 ± 0.07 pmole/10 cells) (p < 0.05). No
inhibiting effect was observed when rBP53 antibody was
incubated with WSSV free cells (Data not shown).
bp53 expression responses to WSSV infection
Time-course analysis of bp53 expression was performed
after WSSV challenge in 24 hours. Expression profile of
bp53 in hemolymph was shown in Figure 5A. The level
Liang et al. Virology Journal (2015) 12:49
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Figure 2 Localization of BP53 in hemocytes by immunofluorescence assay. (A) and (B), Normal shrimp hemocytes incubated with anti-BP53
polyclonal antibody, which showed punctate structures distributed over the entire cell surface. (C), Negative control. Normal shrimp hemocytes
incubated with pre-immune rabbit serum instead of anti-BP53 polyclonal antibody, which had no detectable fluorescence signal. (D), Permeabilized
shrimp hemocytes incubated with anti-BP53 polyclonal antibody, which showed the intracellular expression of BP53 in a characteristic reticular pattern.
(E), Normal hemocytes incubated with anti-actin antibody as control group, which didn’t show any positive signals. (F), Permeabilized cells incubated
with anti-actin antibody, while showed positive actin signals. Evans blue was used to visualize intact cells, and DAPI was used to visualize nuclei.
of bp53 expression dramatically decreased in first 4 h
post injection, slightly increased at 8 h till 18 h, both in
the WSSV challenged and the control group. At 24 h
post injection, the expression of bp53 in the WSSV challenged group decreased significantly (p <0.05) in comparison to the gene expression in control group. The
transcription levels of bp53 in gill showed in Figure 5B.
The level of bp53 expression decreased at 4 h post injection and maintained a low level at 8 h in both groups.
After 12 h post injection, bp53 expression increased till
the end of the experiment in WSSV challenged group,
with significant difference (p <0.05) to the gene expression in control group.
bp53 is essential for shrimp survival
The endogenous genes can be knocked down successfully after 10 h post injection of bp53-specific dsRNA,
and such silencing effect could remain from 33 h up to
5 days post-injection (Figure 6A). Even without WSSV
challenge, over 50% of treated shrimp were died by day
5 post-dsRNA injection (Figure 6B), suggesting that
BP53 expression is essential for shrimp survival.
Discussion
Since the first identification of ATP synthase on the surface of cancer cells in 1994 [12] fewer have attempted to
characterize its function. We have initially identified an
ATPsyn β subunit (named as BP53) as WSSV binding
protein in shrimp Litopenaeus vannamei [8]. However,
the biological functions of ATPsyn β involved in WSSV
infection remain unclear. In this study, we extended our
initial study and elucidated that BP53 was not only localized inside the cells but was also on the entire cell surface of some hematocytes and the cuticular epithelium
membrane of gills, which satisfied the basic requirement
to be a candidate receptor for WSSV. Interestingly, involvement of ATPsyn β in the entry of chikungunya
virus (CHIKV) into insect cells has been recently
Liang et al. Virology Journal (2015) 12:49
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Figure 3 The localization of BP53 in gill secondary filaments. The expression of BP53 was analyzed by immunofluorescence microscopy and
immunogold electron microscopy, respectively. (A) and (B), Gill tissues incubated with anti-BP53 polyclonal antibody, the fluorescence signals
were developed by FITC-conjugated HRP, which showed numerous green spots distributed along the cuticular epithelium of gills. Pre-immune
serum staining was performed as negative control (C), of which no green fluorescence spot was observed. Evansblue was used to visualize gill
tissue, which showed red color under 550 nm laser light. (D), Histological slide of gill tissue observed under microscopy (1000 x), T-E stained. (E),
The framed part of picture D was observed under electron microscopy. (F), Enlarged picture of part of the membrane picture E, which showed
golden particles appeared along the cell membrane of the epithelium under the cuticle of gills. (G), Negative control, pre-immune serum was
used as primary antibody instead of anti-BP53 Ab. Arrows point positive staining.
demonstrated, and showed a significant reduction in
viral entry and virus production by both antibody inhibition and siRNA-mediated down regulation experiments
targeted to ATPsyn β [13]. Our current results revealed
the evidence that the same protein, ATPsyn β, involved
in the infection of WSSV to shrimp, and possibly
CHIKV and dengue virus to insect cells as well [13,14].
These informations will lead us a better illustration of
the entry process of WSSV into the host cells, as well as
the dengue virus into insect cells.
Lin et al. [10] used several different methods to clearly
demonstrate that ATP synthase present on the surface
of Hpt cells and not on mature blood cells (hemocytes)
of crayfish (P. leniusculus). In present study, we demonstrated the presence of ATPsyn β on cytoplasm membrane of gill epithelia and some circulating hemocytes in
L. vannamei. It’s generally agreed that haematopoietic
tissue (HPT) is responsible for production and supply of
the haemocytes. Hemocytes are synthesised and partly
differentiated in the hematopoietic tissue, very little cell
proliferation could occurs in the circulation [15]. Besides
these, there were three types of mature hemocytes and
five immature hemocytes [15-17], so ATPsyn β was possible located on the surface of some of these circulating
cells in shrimp. This result was strengthened by the
localization of ATPsyn β on epithelial cells of shrimp gill
in our study by immunofluorescence assay and immunogold labeling technique. Furthermore, from the viewpoint
of astakine receptor, ATP synthase enzyme complex was
likewise detected only on the surface of HPT cells and not
on any hemocytes in crayfish, because the expression of
crayfish astakine was found to be restricted to the blood
cells lineage [10]. While shrimp astakine mRNA is
expressed in multi-tissues and organs, such as eyestalk,
subcuticular epithelium, gills, heart, hepatopancreas,
lymphoid organ, intestine, muscle, nerve and hematocytes
[18,19]. Thus, there might have some circulating hemocytes with functional characteristics as hematopoietic stem
cell in shrimp, which presented cell surface ATPsyn β.
However, further investigation needed before any postulation on the difference of molecular mechanisms involved
in the hematopoiesis between crayfish and shrimp.
Liang et al. Virology Journal (2015) 12:49
Figure 4 ATP generation on shrimp hemocytes surface measured
by bioluminescent luciferase assay. (1) ATP production from WSSV
free cells, (2) ATP production from cells with WSSV bound on the
surface, (3) ATP production from WSSV naturally infected shrimp
cells, (4) ATP production from WSSV infected cells that incubated
with anti- rBP53-antibody. Asterisk (*) indicates a significant statistical
difference between groups (p < 0.05).
It has been reported that F1FO ATP synthase on endothelial cell surface is actively involving in ATP synthesis
in human tumor, therefore, the generally accepted concept of ATP synthesis as a strict intracellular process
now appears questionable [20]. However, it’s unknown
for the presence of cell surface F1FO ATP synthase and
its roles in ATP synthesis in crustaceans. We speculate
that the whole ATP synthase complex is present on the
cell surface since its core component, the β subunit, has
been confirmed to localize on cell surface. We quantified
a detectable cell surface ATP production in shrimp
hemolymph which indicated that cell surface ATP synthase is active in shrimp. Moreover, a significant suppressive effect on ATP synthesis activity on the cell
surface has been demonstrated after WSSV infection. It’s
well known that WSSV is an extremely virulent pathogen affecting various shrimps with a very rapid breakout
[1]. Such rapid replication of WSSV in host cells will
lead the quickly consuming of host ATPs and impact
other energy-dependent biological function of the host
cells, and eventually result in cell death [21].
On the other hand, the involvement of ATPsyn β, and
its ligand astakine in hematopoiesis have been reported
in crayfish, and in shrimp Penaeus monodon [22,23] and
L. vannamei [19]. In our recently research, we have also
identified astakine in L. vannamei (LvAST) and the envelope protein VP37 of WSSV competitively bound to
BP53. LvAST and WSSV both likely use ATPsyn β on
target cells as a receptor [24]. Given the role of ATPsyn
Page 5 of 10
β involves in hematopoiesis in crayfish [10], WSSV infection lead to host cell death possibly triggered by the
competition between LvAST and the viral protein VP37
in binding to BP53. Further study is needed to clarify
how the cellular process is carried through in host during WSSV infection.
In the time course analysis of bp53 expression in responses to WSSV infection bp53 expression was downregulated in the early time post-injection in both WSSV
challenged shrimp and the control shrimp. The penetration by the needle or virus stimulation appeared to induce some immune reaction, which in turn affected the
normal physiological function of the shrimp, and the expression of bp53 appeared down-regulation. At 8 h till
18 h post WSSV-challenge, the level of bp53 expression
in hemolymph was just slightly increased after dramatically decreased in first 4 h post injection. However, there
was a significantly up-regulation of bp53 gene expression from 12 h to 18 h post WSSV-challenge in shrimp
gills, which might related to a different function of gill
and hemocytes in systemic immune response to the
WSSV. At 24 h post WSSV injection, the expression of
bp53 decreased both in hemolymph and gill, which
might due to death of hemocytes and cells number decreased in WSSV-infected shrimp [25].
Conclusions
Taken together, our results in the present study provide
evidences in support of the hypothesis that ATPsyn β on
host cell surface serves as a receptor for WSSV. Furthermore, the research revealed a possible molecular mechanism during WSSV infection, by which WSSV appears
to disturb the host energy metabolism by interacting
with host ATPsyn β. It also likely indicated the important role of cell surface ATP energy in WSSV’s binding
and infection process.
Materials and methods
Shrimp
L. vannamei (Crustacea, Decapoda), approximately 8 g
(fresh weight) and 6 to 8 cm long, were purchased from
a local shrimp farm in Qingdao, Shandong Province,
China. Shrimp were cultured in 80 L tanks (at 25°C)
filled with air-pumped sea water. These shrimp were free
of WSSV as tested by PCR.
Some naturally infected L. vannamei shrimp with
WSSV were tested by PCR, and collected from a shrimp
farm in Qingdao, Shandong Province, and were used in
the study for cell surface ATP measurements.
Virus source
A WSSV inoculum was prepared from WSSV-infected
shrimp cephalothorax (2.16 × 103 LD50/ml). Frozen infected tissue was homogenized in sterile HOPBS
Liang et al. Virology Journal (2015) 12:49
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Figure 5 Time-course of bp53 expression in gill and hemolymph after WSSV challenge. A relative quantitative real-time PCR assay was
applied to study bp53 differential expression profiles in L. vannamei hemolymph (A) and gill (B) in response to WSSV infection within 24 h. Gene
expression quantification was determined using the 2−ΔΔCt method. Actin was used as an internal control. Error bars indicate standard deviations
(n = 3). Significant differences between the expression level in each time point post injection and the original level were indicated with an
asterisk (p < 0.05).
(288.8 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
1.4 mM KH2PO4) and centrifuged at 500 g for 10 min.
The supernatant was filtered through a 0.45-μm Millipore filter and diluted 106 times which was then used
for inoculation to obtain WSSV-infected shrimp.
The intact WSSV viral particles from infected crayfish
tissues were purified by differential centrifugation as described by Xie et al. [26]. The optical density of the purified sample was measured at 600 nm wavelength using a
spectrophotometer. A formula C (virions/μl) = 3.34 ×
108 × OD600, which established by Zhou et al. [27] was
used to convert the optical density of purified WSSV
preparation into the virion concentration.
BP53 polyclonal antibody preparation and specificity
characterization
The purified recombinant BP53 (rBP53, GenBank accession number EU401720) was produced in E.coli expression
Liang et al. Virology Journal (2015) 12:49
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Figure 6 Effect on shrimp survival after silencing of the bp53 gene. (A), Endogenous bp53 mRNA expression after dsRNA injection. BP53
group: bp53-specific dsRNA injection; GFP group: gfp-specific dsRNA injection. Representative gels of RT-PCR products of bp53 (482 bp) and ef
(187 bp, internal control) mRNAs from hemolymph collected at different time points. Label 2 log M indicates the 0.1-10.0 kb DNA ladder marker,
while h = hours and D = days post-injection. (B), The cumulative mortality after dsRNA injection without WSSV challenge. BP53 group: bp53-specific
dsRNA injection; GFP group: gfp-specific dsRNA injection; NaCl group: NaCl injection instead of any dsRNA.
system according to the method described previously [8],
and was used as an antigen. A healthy New Zealand rabbit
was subcutaneously injected with 200 μg purified rBP53 in
1 ml 0.9% NaCl as the primary immunization. Two additional booster injections were performed every 4 weeks.
Ten days after the third injection, blood was collected from
the immunized animal by exsanguination under general
anesthesia and kept at room temperature for 3 h and then
centrifuged for 10 min at 300 g to eliminate microclots and
lipids. Serum was then collected and stored at-20°C. Preimmune blood was collected from the ear artery of the
same animal as control. The antiserum was incubated with
lysates from E. coli Top10 cells with blank plasmid pBADgIIIB, which is produced by our lab, prior to applying for
western blot and localization assays.
For the antibody specificity test, a western blot was
carried out. Firstly, the lysates from E. coli Top10 cells
with blank plasmid pBAD-gIIIB (as negative control), lysates of E. coli Top10 cells with recombinant plasmid
pBAD-gIIIB-BP53 and gill membrane proteins extracted
from WSSV-free L. vannamei were subjected to 12%
SDS-PAGE, then transferred to PVDF membranes. The
membrane was blocked with 10% BSA in PBS buffer
containing 0.05% Tween 20 for 2 h at 37°C. Followed,
1:5000 rabbit anti-rBP53 antibody was added and incubated for 2 h at 37°C. After three washes with PBS contained 0.05% Tween 20, the membrane was incubated
with 1:6000 horseradish peroxidase-conjugated goat
anti-rabbit antibody (Invitrogen, USA) at 37°C for 1 hour.
After wash, the signal was generated by BCIP/NBT substrate kit (Pierce, USA).
Localization of BP53 in hemocytes by
immunofluorescence assay
Hemolymph was collected from three WSSV-free L.
vannamei shrimp using a 1-ml tuberculin syringe with a
26-gauge needle, 3.8% sodium citrate used as anticoagulant. Then, hemocytes were collected immediately by
centrifugation at 600 g for 10 min, and suspended in 2 X
L15 medium (GIBCO, China) with 20% (vol/vol) fetal
calf serum (FCS) and 50 μg/ml antibiotics (10000U/ml
Penicillin-10 mg/ml Streptomycin Solution). Then the
hemocytes were incubated at 28°C for 1 h to allow cells
to attach to the bottom of a petri dish. The hemocytes
were then washed with PBS, divided into two groups
and used for the following process. In control group,
cells were permeabilized in absolute ethanol at RT for
3 min. All the cells were incubated with 10% bovine
Liang et al. Virology Journal (2015) 12:49
serum VI (Sigma) in PBS for 2 h and washed before incubation with rabbit anti-BP53 antibody (1:200) or preimmune rabbit serum (1:200, negative control) at 28°C
for 2 h. To test whether cells were permeabilized in each
treatment, the cells were incubated with the antibody of
cytoskeleton protein marker, actin, as the control. All
cells were washed, and incubated with FITC-conjugated
goat anti-rabbit IgG antibody (1:400, Invitrogen) and
0.01% (vol/vol) Evansblue for 1 h at 28°C. The nuclei
were stained with DAPI. After final washes, cells were
visualized under a confocal laser scanning microscopy
(Nikon A1, Japan) at 425 nm and 488 nm wavelength
correspondingly.
Subcellular localization of BP53 in L. vannamei gills
Immunofluorescence assay
Gill secondary filaments tissues were removed from
WSSV-free shrimp L. vannamei and immediately fixed
in 4% formaldehyde fixation buffer for 12 h. The ultrathin paraffin sections were prepared and mounted on
slides with coated poly-L-lysine. After de-waxing, sections were treated in 3% (vol/vol) methanol-H2O2 for
20 min, followed by high-pressure antigen retrieval in
0.5 M EDTA (pH 8.0) for 10 min. The indirect immunofluorescence assay was performed following the same
procedure as described above.
Immunogold labeling technique
The gill filaments tissues were removed from WSSV-free
L. vannamei and immediately fixed in glutaraldehydeosmium acid buffer. The tissues were then embedded in
Spurr. Sectuibs (50 nm thick) were collected on carboncoated nickel grids. Sections were incubated with 1%
H2O2 for 30 min followed by washing in distilled H2O
and 0.05% TBS-Tween 20 three times (5 min per wash).
Non-antigenic sites were blocked by incubating with 1%
(wt/vol) TBS-BSA for 2 h at 37°C. The grids were then
incubated with 200 μg/ml rabbit anti-rBP53 antibody in
blocking buffer at 37°C for 1 h. After three washes, the
sections were incubated with 10-nm colloidal goldconjugated goat anti-rabbit IgG (Sigma, USA) in 1:200
dilution with blocking buffer for 1 h at 37°C. The sections were then washed three times with TBST buffer,
followed three washes with H2O. Then the sections were
post-stained with 2% (vol/vol) aqueous uranyl acetate for
10 min at room temperature, and incubated with Pb citrate for an additional 2 min. Parallel controls were performed with pre-immune serum. The sections were
observed under an electron microscope (JEOL JEM1200EX, US).
Cell surface ATP assay
The cell surface ATP assay were performed as described
with modifications [20]. Hemocytes were collected by
Page 8 of 10
centrifuging WSSV-free or WSSV naturally infected
shrimp hemolymph, which was a mix of 3 individuals in
each group, at 600 g for 10 min with 3.8% sodium citrate
as anticoagulant. Hemocytes were seeded into 24-well
plates at a density of 1000 cells per well and cultured in
2 X L15 medium with 20% FCS at 28°C for 3 h. Cells
from WSSV-free shrimp were divided into two experimental groups. One group was incubated with purified
WSSV at 4°C for 1 h, which allowed the virus just bound
on the cells surface and not go inside the cells. One
group was used for the following measurement without
WSSV incubation. All the cells above were washed by
Hepes buffer (10 mM Hepes, 150 mM NaCl, pH7.4),
and incubated with 0.3 ml Hepes buffer containing
200 μM ADP, 20 mM potassium phosphate (Pi) and
2 mM MgCl2 at room temperature for 3 min. Supernatants were collected and centrifuged before assaying for
ATP production by bioluminescent luciferase assay.
Quantification of cell-surface ATP by bioluminescent
luciferase assay
Aliquots of 100 μl supernatants from cell surface ATP
assays as described above were analyzed using an ATP
bioluminescence assay kit (Beyotime, China). In this
study, only ATP is readily detected by the specific enzymatic reaction [20]. ATP generation was quantified by
Varioskan flash multimode reader (Thermo scientific,
Finland) and signals were recorded as RLU value. Data
are expressed as picomoles of ATP produced per 10 cells
relative to the standard determined under the same conditions with each experiment. All the experiments were
carried out in triplicates, values reported are means of
three replicates ± SD. One way ANOVA tests were used
to test for statistically significant differences (p < 0.05).
bp53 expression upon WSSV infection by relative
quantitative real-time PCR assay
bp53 differential expression profiles in L. vannamei
hemolymph and gill in response to WSSV infection was
analyzed by relative quantitative real-time PCR. Shrimp
was injected with 100 μL WSSV inoculum on the lateral
side in the experimental group or with 100 μL sterile
HOPBS in the control group with a 1-ml sterile syringe.
Hemolymph and gill tissue from six shrimp per group
per time point, were collected at 0 h, 2 h, 4 h, 8 h, 12 h,
18 h and 24 h post inoculation for RNA extraction.
Total RNA was extracted from the hemolymph using
TRI Reagent (Invitrogen, USA) following the manufacturer’s instructions. 2 μg RNA was reversely transcribed
with random 6 mers primer and the oligo (dT) primer
using M-MLV reverse transcriptase (NEB, New Finland)
to obtain first-strand cDNA. A 171-bp bp53 gene fragment was amplified using primers 5’-TCT CTC TGA
AGG ATG ATA C-3’ and 5’-GTG TGA AGC GGA
Liang et al. Virology Journal (2015) 12:49
AAA T-3’. Relative transcript quantities were calculated
using the 2−ΔΔCt method as described by Livak and
Schmittgen [28] with β-actin as the reference gene amplified from the same samples. A 170-bp β-actin gene
fragment was amplified using specific primers 5’
CGACCTCACAGACTACC 3’ and 5’ AGGACTTCTCCAGCG 3’. The PCR program was 95°C for 10 seconds
followed by 40 cycles of 95°C for 6 s, 51°C for 30 s, and
72°C for 20 s. The real-time RT-PCR was performed in
triplicate for the same sample in each experimental
group. One way ANOVA tests were used to test for statistically significant differences (p < 0.05).
RNAi-mediated silencing of the bp53 gene
Double-stranded (ds) RNA corresponding to the bp53
sequences was synthesized by in vitro transcription using
the commercial RiboMAX™ T7 Express System kit (Promega, USA). Sense and antisense DNA templates for
in vitro transcription were generated by PCR. The forward primers for both DNA templates were designed to
contain T7 promoter sequence at the 5’ end (indicated
by italics). The primers used for amplification of the
sense DNA template were 5’ GAT CCT AAT ACG ACT
CAC TAT AGG CCA TCT ATG TAC CTG CTG ATG
ACT T 3’ and 5’ GCA GCC AAC TGT TCT GCC TTT
TC 3’. The primers used for amplification of the antisense DNA template were 5’ GGC CAT CTA TGT ACC
TGC TGA TGA CTT 3’ and 5’ GGA TCC TAA TAC
GAC TCA CTA TAG CAG CCA ACT GTT CTG CCT
TTT C 3’. Plasmid pBAD-gIIIA-BP53, which encodes
the full-length cDNA of the bp53 gene, was used as a
template for PCR. An unrelated dsRNA corresponding
to the green fluorescence protein (GFP) gene was prepared as a control as previously described [29].
Test shrimp (30 shrimp per group) were injected
intramuscularly in the fourth or fifth abdominal segment
with bp53-specific dsRNA (20 μg) or GFP-specific
dsRNA (20 μg) dissolved in 100 μl of 150 mM NaCl solution using a 1-ml tuberculin syringe with a 26-gauge
needle. Control shrimp were injected with 150 mM
NaCl. Hemolymph (200 μl) was collected from three
shrimp into 200 μl pre-cooled anti-coagulant solution
(AC-l) (0.45 M NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citric acid, 10 mM EDTA, pH 4.6) [30] at
0 h, 10 h, 24 h, 33 h, and day 2 to day 5.
RT-PCR analysis for bp53 knock down
Total RNA was extracted from the hemolymph using
TRI Reagent (Invitrogen, USA) following the manufacturer’s instructions. RNA was quantified by spectrophotometer, and 100 ng RNA of each sample was used for
one-tube RT-PCR (Roche, USA). For bp53 gene, the following primers were used: 5’ ATT TCT TTC CAG AGC
CCT G 3’ and 5’ GGTATTGCCGAGTTGGGT 3’.
Page 9 of 10
Elongation factor (EF) was used as an internal control
with the following primers (5’ GGT GCT GGA CAA
GCT GAA GGC 3’ and 5’ CGT TCC GGT GAT CAT
GTT CTT GAT G 3’). The PCR conditions for bp53 and
ef were as follows: 50°C for 30 min, 94°C for 2 min,
followed by 28 cycles of 94°C for 10 s, 55°C for 30 s, and
68°C for 45 s, with a final extension at 68°C for 7 min.
The PCR products of bp53 (482 bp) and ef (187 bp) were
analyzed by 1.2% agarose gel electrophoresis.
Abbreviations
WSSV: White spot syndrome virus; VOPBA: Virus overlay proteins binding assay.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YL conceived of the study, participated in its design and coordination,
carried out localization studies, cell surface assay, RNAi, data analysis, and
drafted the manuscript. MLX participated in the localization assay. XWW and
XXG participated in the cell surface ATP assay. JJC carried out the RT-PCR. CL
participated in the localization assay. JH contributed in revising the manuscript
and coordination. All authors read and approved the final manuscript.
Acknowledgments
This study was supported by the project under the National Natural Science
Foundation of China (Grant No. 31101934), the National Basic Research
Program of China (973 Program, Grant No. 2012CB114400), and the Special
Fund of the Construction Program for “the TAISHAN Scholar”. The authors
would like to thank Ms. Ying-Yi Hou and Mr. Jin-Shan Tan in the College of
Medical Science, Qingdao University for their kind assistance in the electron
microscopic preparation. The authors would like to thank Dr. Jun Li in Lake
Superior State University and Dr. Qing-Gang Xue in Louisiana State University
for their suggestions in revising this paper.
With this paper, we would like to commemorate Jun-Jun Cheng, who loved
scientific research but died at an early age.
Received: 29 October 2014 Accepted: 9 March 2015
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